Immunostimulatory compositions and methods of use thereof

ABSTRACT

Immunostimulatory compositions and methods of use are described to either enhance or diminish the immune stimulation effects of a honey or honey isolate by recognition of the presence of type II arabinogalactan compounds and utilizing this knowledge to tailor the concentration of such compounds thereby adjusting the immune stimulation effects.

RELATED APPLICATIONS

This application claims priority from NZ585118 dated 5 May 2010, thecontents of which are incorporated herein by reference.

BACKGROUND ART

Honey used in wound dressings has been extensively discussed and taughtin the art.

The applicants co-pending patent application, PCT/NZ2009/000232 teachesabout the recent finding that honey in the context of wound dressingsheals a wound via three distinct phases being an antimicrobial phase, animmune stimulation phase and an anti-inflammatory phase. As noted inthis earlier application, the different phases may occur one after theother or in conjunction with one another, particularly in the case ofthe antimicrobial and immune stimulation phases. These distinct phasesdo however appear to be critical in providing the particular woundhealing characteristics observed in honey.

This application is primarily centered on the immune stimulation orpro-inflammation phase of healing.

Inflammation is typically considered a negative or to be avoided actionin the context of wound healing—i.e. why would you inflame an alreadyinflamed wound? The applicants have found that inflammation at least inthe honey wound healing context is in fact beneficial for most woundapplications contrary to that expected, particularly when placed incontext with the other phases of healing. As well as and distinct toanti-microbial effects, honey appears to prime or kick start the immunesystem into action via this second phase of healing, a characteristicnot uncommon in some contexts with positive outcomes e.g. to addresschronic or recalcitrant infections where the natural wound healingprocess has stalled for some reason or alternatively, to prompt apositive reaction such as that observed when probiotic bacteria areintroduced to the gut. Many studies have also been produced showing howhumans and animals such as mice primed via an immune stimulatorychallenge often survive another microbial challenge better than mice notprimed. The actual compound(s) responsible for the inflammatory effectsare to the applicant's knowledge not described in the art.

Arabinogalactan (AG) is a biopolymer consisting of arabinose andgalactose monosaccharides. Two classes exist in nature being plantarabinogalactans and microbial arabinogalactans.

In plants, AG is a major constituent of many gums including gum arabic,gum gutti and so on.

AG is also found in Echinacea and other plant matter, typically in theamount of 0.1% weight or 100 μg/ml-200 μg/ml.

AG may be attached to proteins and the resulting arabinogalactan protein(AGP) functions as a signaling molecule between cells.

It should be appreciated from the above that it would be useful to havean identified immune-stimulant compound that may be isolated or selectedfor when tailoring a composition for medical applications such astopical formulations and which can be manipulated to be used in both thecontext of treating very sensitive wounds through to chronic orrecalcitrant wounds. It is an object of the present to address theforegoing problems or at least to provide the public with a usefulchoice.

All references, including any patents or patent applications cited inthis specification are hereby incorporated by reference. No admission ismade that any reference constitutes prior art. The discussion of thereferences states what their authors assert, and the applicants reservethe right to challenge the accuracy and pertinence of the citeddocuments. It will be clearly understood that, although a number ofprior art publications are referred to herein, this reference does notconstitute an admission that any of these documents form part of thecommon general knowledge in the art, in New Zealand or in any othercountry.

It is acknowledged that the term ‘comprise’ may, under varyingjurisdictions, be attributed with either an exclusive or an inclusivemeaning. For the purpose of this specification, and unless otherwisenoted, the term ‘comprise’ shall have an inclusive meaning—i.e. that itwill be taken to mean an inclusion of not only the listed components itdirectly references, but also other non-specified components orelements. This rationale will also be used when the term ‘comprised’ or‘comprising’ is used in relation to one or more steps in a method orprocess.

Further aspects and advantages will become apparent from the ensuingdescription that is given by way of example only.

SUMMARY

The application broadly relates to the discovery that honey,particularly honey with non-peroxide antibacterial activity, containstype II arabinogalactan (AG) compounds that act to stimulate or primethe immune system. Based on this knowledge, topical formulations such aswound dressings may be produced to accentuate the pro-inflammatoryeffect or down regulate the pro-inflammatory effect by altering theamount of AG present. Further, the applicants have also identified asynergy in immune stimulation when honey is fortified with AG.

In some embodiments there is provided a honey isolate containing type IIarabinogalactan (AG).

In some embodiments there is provided a honey analogue including a honeyisolate containing type II arabinogalactan (AG).

In some embodiments there is provided a honey fortified with a honeyisolate containing type II arabinogalactan (AG).

In some embodiments there is provided a composition formulated fortopical application to a body area of a patient in need thereofcontaining:

-   -   (a) a honey isolate containing type II arabinogalactan (AG); or    -   (b) a honey analogue including a honey isolate containing type        II arabinogalactan (AG); or    -   (c) a honey fortified with a honey isolate containing type II        arabinogalactan (AG); or    -   (d) combinations of the above.

In some embodiments there is provided a method of stimulating the immunesystem of a patient in need thereof by topical application of acomposition to a body area of the patient wherein the compositioncontains:

-   -   (a) a honey isolate containing type II arabinogalactan (AG); or    -   (b) a honey analogue containing a honey isolate containing type        II arabinogalactan (AG); or    -   (c) a honey fortified with a honey isolate containing type II        arabinogalactan (AG); or    -   (d) combinations of the above.

In some embodiments there is provided the use of a composition in themanufacture of a composition formulated for topical application forstimulating the immune system of a body area on a patient in needthereof wherein the composition contains:

-   -   (a) a honey isolate containing type II arabinogalactan (AG); or    -   (b) a honey analogue including a honey isolate containing type        II arabinogalactan (AG); or    -   (c) a honey fortified with a honey isolate containing type II        arabinogalactan (AG); or    -   (d) combinations of the above.

In some embodiments there is provided a method of minimising theimmune-stimulatory effects of a honey based topical composition by thestep of removing type II arabinogalactan (AG) from the honey in thecomposition.

In some embodiments there is provided the use of a honey based topicalcomposition with type II arabinogalactan (AG) removed from the honey inthe manufacture of a topically administered composition for minimisingstimulation of the immune system of a body area on a patient in needthereof.

In some embodiments there is provided a method of treatment of arecalcitrant skin condition on a topical body area on a subject in needthereof by:

-   -   a. application of a composition initially that includes:        -   I. a honey isolate containing type II arabinogalactan (AG);            or        -   II. a honey analogue including a honey isolate containing            type II arabinogalactan (AG); or        -   III. a honey fortified with a honey isolate containing type            arabinogalactan (AG); or        -   IV. combinations of the above;            and, as skin healing progresses;    -   b. applying a honey or honey analogue based composition with        type II arabinogalactan (AG) removed from the honey or analogue.

In some embodiments there is provided a method of treatment of asensitive skin condition on a topical body area on a subject in needthereof by:

-   -   a. applying a honey or honey analogue based composition with        type II arabinogalactan (AG) removed from the honey or analogue:        and as skin healing progresses;    -   b. application of a composition initially that includes:        -   I. a honey isolate containing type II arabinogalactan (AG);            or        -   II. a honey analogue including a honey isolate containing            type II arabinogalactan (AG); or        -   III. a honey fortified with a honey isolate containing type            II arabinogalactan (AG); or        -   IV. combinations of the above.

In some embodiments, there is provided a hod of producing a honey basedimmune-stimulatory composition by the step of:

-   -   a. testing a series of honey batches for the presence of type II        AG compounds;    -   b. selecting and blending together honeys based on the test        results to form a honey blend with type II AG levels greater        than 5 μg/g.

In some embodiments there is provided a method of producing a honeybased composition that minimises immune-stimulatory effects by:

-   -   a. testing a series of honey batches for the presence of type II        AG compounds;    -   b. selecting and blending together honeys based on the test        results sufficient to leave less than 5 μg/g type II AG        compounds in the honey blend.

In some embodiments there is provided a method of determining theimmunostimulatory properties of a honey or honey analogue by measuringthe content of type II AG in the honey or honey analogue and wherein:

-   -   a. if the amount of type II AG in the honey or honey analogue is        greater than approximately 5 μg/g, a measurable        immunostimulatory response is predicted; and,    -   b. if the amount of type II AG in the honey or honey analogue is        less than approximately 5 μg/g, a measurable immunostimulatory        response is not predicted.

In some embodiments there is provided a method of producing an isolateof the type II AG compounds from honey by obtaining a honey and thenprocessing the honey by steps selected from filtration,ultra-filtration, reverse osmosis, solvent extraction, precipitation, orcombinations thereof and collecting a high molecular weight isolate fromthe processing step.

BRIEF DESCRIPTION OF THE DRAWINGS

Further aspects will become apparent from the following description thatis given by way of example only and with reference to the accompanyingdrawings in which:

FIG. 1 illustrates a structure of honey-based type II AG compounds;

FIG. 2 illustrates the effect of toxicity (cell survival) of THP-1cells. Cells were treated with in the absence (⋄) and presence (□) ofCamptothecin (A) and in the absence (⋄) and presence (□) of artificialhoney solution (B). Data mean of two replicates and the bars displaysthe standard error (SEM);

FIG. 3 illustrates the effects of 21 honey samples on cells (A, INTPH 1and 3: B, 05 and 08; C, 07 and 07; D 10 and 12; E, 14 and 15; F, 16 and17, G, 18 and 19 and H, 20 and 21 (♦ and ▪, respectively)) with measuredeffects (solid line) compared to standardized effects (relative to sugarcontrol ▴) (dashed line). Data mean of two replicates. Standard errorsare not shown;

FIG. 4 illustrates the cytoxicity effects of all 18 honey samples (5%(▪) expressed relative to negative control (100%) (−) and positivecontrol (10 μM carnptothecin) (▪) Data mean of two replicates and thebars displays the standard error (SEM);

FIG. 5 illustrates five proton NMR spectra of (A) H01, (B) H03, (C) H15,(D) H16 and (E) H20;

FIG. 6 illustrates size exclusion chromatography (SEC) profiles of highmolecular weight honey fractions;

FIG. 7 illustrates results for a trial testing the pro-inflammatoryresponse attributable to arabinogalactans where H=honey; AH=artificialhoney; AG=arabinogalactans (type 1); AG=Arabinogalactan concentrationsfrom 2.5% to 1% which is equivalent to honey concentration (i.e. 10% ofhoney contains 8 μg of AG, 2.5% contains 2 μg, 2%=1.6 μg, 1.5%=1.5 μgand 1%=1 μg of AG);

FIG. 8 illustrates a graph showing cytokine production of THP-1 cellline when contacted with various honeys, a honey analogue and MGO;

FIG. 9 illustrates a graph showing cytokine production of THP-1 cellline when contacted with varying size kanuka fractions;

FIG. 10 illustrates a graph showing cytokine production of THP-1 cellline when contacted with varying compositions with and without AGP;

FIG. 11 illustrates a graph illustrating cytokine production as relatedto dose of AGP isolate.

DETAILED DESCRIPTION

As noted above, the application broadly relates to the discovery that AGcompounds are present in honey and, that these compounds are associatedwith a secondary phase of healing being to stimulate the immune systemof the subject to whom the medical formulation is applied. Also, asnoted above, the applicants have also identified a synergy in immunestimulation when honey is fortified with AG.

For the purposes of this specification, the term ‘arabinogalactan’ or‘AG’ or grammatical variations thereof refers to biopolymers containingarabinose and galactose monosaccharides.

The term ‘type II’ when used in reference to AG compounds refers to afamily of highly branched polysaccharide compounds rich in galactose andarabinose. They consist of a (1-3)-β-D-galactan backbone having(1-6)-β-D-galactan side chains, which in turn are modified by arabinose.Short arabinose oligosaccharide chains may additionally decorate thegalactan backbone.

The term ‘arabinogalactan protein’ or AGP or grammatical variationsthereof refers to arabinogalactan compounds where the polysaccharideunits are attached to multiple sites on a core protein, rich inhydroxyproline.

The terms ‘unique manuka factor’, ‘UMF’, ‘non-peroxide activity’,‘methylglyoxal’ or ‘MGO’ all refer to the anti-microbial activity of ahoney above that attributable to the antimicrobial effects of honey pHand osmolarity.

The term ‘honey’ refers to honey produced either predominantly from onebotanical species (monofloral honey) or produced from multiple speciesand/or mixtures of honey (multifloral honey).

The term ‘honey analogue’ or grammatical variations thereof refers to asugar syrup solution that approximates the basic chemical and/orphysical properties of natural honey being made up of glucose, fructose,water and hydrogen peroxide and/or glucose peroxidase enzyme.

The term ‘isolate’ or grammatical variations thereof refers to acomposition containing a purified concentration of type II AG compoundsseparated from a honey as produced in nature or separated from a honeyfraction such as a honey UMF fraction or similar.

The term ‘secondary phase of healing’ refers to an immune stimulantaction characterised by the production of inflammatory cytokinesincluding but not limited to TNFα, IL-6 and IL-10.

The term ‘wound dressing’ refers to a dressing adapted for applicationto a topical wound containing honey and/or a honey derivative.

The term ‘topical’ refers to placement on a body area of a subject suchas skin as well as mucosal areas such as the oral cavity e.g. gums, thenasal cavity and the vaginal cavity. The term may also encompass theintestine wall owing to the fact that type II AG compounds arecomparatively stable and on oral delivery would reach the intestineschemically intact.

The terms ‘chronic’ or ‘recalcitrant’ are used interchangeably to referto a skin area or broken skin area such as a burn or wound that iseither not healing or is only healing slowly despite treatment. Thisstyle of healing may be characterised by little macrophage activity ator around the skin area.

The term ‘sensitive’ or grammatical variations thereof refers to a skinarea that the subject finds particularly painful.

The terms ‘immunostimulatory’, ‘stimulate’, ‘pro-inflammatory’ orgrammatical variations thereof refer to the subject's immune systembeing activated to the extent that macrophage cells are present at thewound site and produce cytokines consistent with an inflammatoryresponse including but not limited to TNFα, IL-6 and IL-10.

The terms ‘prime the immune system’ and/or ‘stimulate the immune system’refer to the presence of macrophage cells producing or capable ofproducing inflammatory related cytokines.

The term ‘fortify’ or grammatical variations thereof refers to additionof a separate component to a base material and/or the addition of aseparate component to increase the concentration of the componentalready in the base material.

In some embodiments there is provided a honey isolate containing type IIarabinogalactan (AG).

The average molecular weight of the AG in the isolate may be from 40,000to 1,100,000.

The ratio of arabinose to galactose may range from 0.1 to 2.0 partsarabinose to 1.0 parts galactose. The ratio may be 0.65 to 1.21arabinose to 1.0 parts galactose.

The isolate may include type II AG with a structure as shown in FIG. 1.

The isolate may include greater than 5 μg/g type II AG or sufficienttype II AG to cause significant immunostimulatory effects in monocytes.The isolate may include greater than 10 μg/g type AG.

The honey from which the isolate is derived may be selected from honeysderived form the plant genus Leptospermum, Kunzea, Weinmannia, Knightia,Metrosideros, Fagus, Trifolium, Myrtaceae, and combinations thereof. Inselected embodiments, the honey may of manuka origin. The honey may beof kanuka origin. The honey may be of clover origin. The honey mayinstead be a multifloral honey.

In some embodiments there is provided a honey analogue including a honeyisolate containing type II arabinogalactan (AG). The analogue mayinclude greater than 10 μg/g type II AG.

In some embodiments there is provided a honey fortified with a honeyisolate containing type arabinogalactan (AG). The honey may be fortifiedsufficient to increase the concentration of type II AG to greater than 5μg/g. The concentration may be greater than 10 μg/g.

In some embodiments there is provided a composition formulated fortopical application to a body area of a patient in need thereofcontaining:

-   -   (a) a honey isolate containing type II arabinogalactan (AG); or    -   (b) a honey analogue including a honey isolate containing type        II arabinogalactan (AG); or    -   (c) a honey fortified with a honey isolate containing type II        arabinogalactan (AG); or    -   (d) combinations of the above.

The composition as described above wherein the patient may be a human.Alternatively, the patient may be a non-human animal.

The composition described above may be formulated as: a dressing, acream, an ointment, a gel, and combinations thereof.

The type II AG compound(s) described above may be arabinogalactanprotein (AGP) compounds.

The composition may include greater than 5 μg/g AG compounds or mayinclude greater than 10 μg/g AG compounds.

In the above embodiments, a non-honey based AG containing compositionmay be added to the composition. For example a plant based AG compoundisolate may be added. Synthetically produced AG compounds may be added.A compound including AG may also be added e.g. grass or an extractthereof.

In some embodiments there is provided a method of stimulating the immunesystem of a patient in need thereof by topical application of acomposition to a body area of the patient wherein the compositioncontains:

-   -   (a) a honey isolate containing type II arabinogalactan (AG); or    -   (b) a honey analogue containing a honey isolate containing type        II arabinogalactan (AG); or    -   (c) a honey fortified with a honey isolate containing type II        arabinogalactan (AG); or    -   (d) combinations of the above.

In some embodiments there is provided the use of a composition in themanufacture of a composition formulated for topical application forstimulating the immune system of a body area on a patient in needthereof wherein the composition contains:

-   -   (a) a honey isolate containing type II arabinogalactan (AG); or    -   (b) a honey analogue containing a honey isolate containing type        II arabinogalactan (AG); or    -   (c) a honey fortified with a honey isolate containing type II        arabinogalactan (AG); or    -   (d) combinations of the above.

In the method or use above, the composition may be formulated forapplication to a recalcitrant topical body area on a patient.

In some embodiments there is provided a method of minimising theimmune-stimulatory effects of a honey based topical composition by thestep of removing type II arabinogalactan (AG) from the honey in thecomposition.

In some embodiments there is provided the use of a honey based topicalcomposition with type II arabinogalactan (AG) removed from the honey inthe manufacture of a composition formulated for topical application forminimising stimulation of the immune system of a body area on a patientin need thereof.

In the above method or use, the composition may be formulated forapplication to a sensitive topical body area on a patient. It shouldalso be noted that the honey based topical composition still providesanti-microbial effects associated with honey even with the isolateremoved i.e. anti-microbial effects due to honey are distinct to immunestimulation effects and caused by different components in honey.

In some embodiments of the above method or use, the concentration oftype II AG in the honey is less than 5 μg/g or a level that yields no orminimal immunostimulatory effects on monocytes. The level may be lessthan 1 μg/g type II AG.

As should be appreciated, the above embodiments approach theunderstanding of the importance of AG in inflammation in two differentways. The immune stimulating embodiments seek to provide a topicalformulation that may be applied to skin areas that are not progressingin healing or where only very slow wound healing is occurring. In thiscontext, the AG content in the dressing is maximised. The converse istrue in the immune stimulation minimizing embodiments where aninflammatory response may cause considerable pain and discomfort to thesubject, in these embodiments the aim is to reduce the content of AG andthereby minimise the inflammatory response while letting other phases ofaction e.g. anti-microbial and anti-inflammatory provide the primaryhealing action.

Further, in the above methods and uses, the patient may be a human.Alternatively, the patient may be a non-human animal.

In some embodiments there is provided a method of treatment of arecalcitrant skin condition on a topical body area on a subject in needthereof by:

-   -   a. application of a composition initially that includes:        -   I. a honey isolate containing type II arabinogalactan (AG);            or        -   II. a honey analogue including a honey isolate containing            type II arabinogalactan (AG); or        -   III. a honey fortified with a honey isolate containing type            arabinogalactan (AG); or        -   IV. combinations of the above:            and, as skin healing progresses;    -   b. applying a honey or honey analogue based composition with        type II arabinogalactan (AG) removed from the honey or analogue.

In some embodiments there is provided a method of treatment of asensitive skin condition on a topical body area on a subject in needthereof by:

-   -   a. applying a honey or honey analogue based composition with        type II arabinogalactan (AG) removed from the honey or analogue;        and as skin healing progresses;    -   b. application of a composition initially that includes:        -   I. a honey isolate containing greater than or equal to 5            μg/g type II arabinogalactan (AG); or        -   II. a honey analogue including a honey isolate containing            type II arabinogalactan (AG); or        -   II. a honey fortified with a honey isolate containing type            II arabinogalactan (AG); or        -   IV. combinations of the above.

Uses of the above honey or honey analogue based composition with anisolate removed as well as those containing isolates may also be in themanufacture a topical preparation method for the treatment of arecalcitrant or sensitive skin area.

Applying the above to a wound example, the method of treatment mayentail initial application or a dressing with pro-inflammatoryproperties to a new and sensitive wound and then, once the initialhealing process begins, removal of the dressing and replacement with adressing that has inflammatory properties to enhance or speed up thewound healing progression. The opposite may also be the case where thewound is recalcitrant so a pro-inflammatory dressing is applied firstand then, once healing progresses, a dressing without pro-inflammatoryeffects may be applied.

As noted above, the immunostimulatory composition may contain greaterthan 5 μg/g or greater than 10 μg/g type II AG compounds. Alternatively,if non-immunostimulatory composition may contain less than 5 μg/g orless than 1 μg/g type II AG compounds.

Further, in the above methods and uses, the patient may be a human.Alternatively, the patient may be a non-human animal.

In some embodiments, there is provided a method of producing a honeybased immune-stimulatory composition by the step of:

-   -   a. testing a series of honey batches for the presence of type II        AG compounds;    -   b. selecting and blending together honeys based on the test        results to form a honey blend with type II AG levels greater        than 5 μg/g.

In the above method, the honey may include kanuka honey.

The honey tested may be produced from a plant or plants selected and/orbred to maximise the level of type II AG compounds in the plant nectarfrom which the honey is produced. The plant nectar may be selected fromthe genus: Leptospermum, Kunzea, Weinmannia, Knightia, Metrosideros,Fagus, Trifolium, Myrtaceae, and combinations thereof.

Optionally, the composition may include further non-honey basedarabinogalactan compounds. For example, a plant based AG compoundisolate may be added. Synthetically produced AG compounds may be added.A compound including AG may also be added e.g. grass or an extractthereof.

In some embodiments there is provided a method of producing a honeybased composition that minimises immune-stimulatory effects by:

-   -   a. testing a series of honey batches for the presence of type II        AG compounds;    -   b. selecting and blending together honeys based on the test        results sufficient to leave less than 5 μg/g type II AG        compounds in the honey blend.

In the above method the honey tested may be produced from a plant orplants selected and/or bred to minimise the level of type II AGcompounds in the plant nectar from which the honey is produced. Plantnectar may be selected from the genus: Leptospermum, Kunzea, Weinmannia,Knightia, Metrosideros, Fagus, Trifolium, Myrtaceae, and combinationsthereof.

In the above methods, the type II AG compound may be AGP.

By way of example, the honeys used in the above embodiments may beselected from honey sources that do not have high levels of AG such asclover honey to make a low AG dressing. Alternatively, high AGcontaining honeys such as kanuka honeys may be selected to produce ahigh AG content dressing.

Further, the natural process of producing honey involves bees collectingpollen from flowering plants that is then converted to honey via thebees in the hive. Besides direct manipulation as noted in earlierembodiments of the honey or use of isolates, an alternative may be toalter the plant source of the honey. For example, as noted aboveLeptospermum or Kunzea genus trees such as manuka or kanuka trees may beselected and bred to produce higher levels of AG in the plant nectarthat the bees then collect and manufacture honey from. Alternatively,plant species (not necessarily from Leptospermum or Kunzea genus) may beselected for their AG production levels in nectar and the hive placedadjacent these species around flowering in order to concentrate the AGlevels in the honey produced by the bees. Examples of alternativespecies may include Weinmannia, Knightia, Metrosideros, Fagus, Trifoliumor Myrtaceae genus plants.

In some embodiments there is provided a method of determining theimmunostimulatory properties of a honey or honey analogue by measuringthe content of type II AG in the honey or honey analogue and wherein:

-   -   a. if the amount of type II AG in the honey or honey analogue is        greater than approximately 5 μg/g, a measurable        immunostimulatory response is predicted; and,    -   b. if the amount of type II AG in the honey or honey analogue is        less than approximately 5 μg/g, a measurable immunostimulatory        response is not predicted.

In the above method, an immune-stimulatory topical formulation may beproduced from the honey or honey analogue if the measured type AG isgreater than 5 μg/g. The amount may be greater than 10 μg/g.

In the above method, a non-immune stimulatory topical formulation may beproduced from the honey or honey analogue if the measured type II AG isless than 5 μg/g. The amount may be less than 1 μg/g.

In the above method, testing may be completed utilising reactions thatinvolve binding type AG compounds with specific antibodies. Optionally,one method may be to utilise Enzyme Linked Immuno Sorbent Assay (ELISA)analysis. ELISA may be a useful detection protocol as it is easilyimplemented in a QA laboratory, can be used to process many samples atonce (40-80), provides accurate results and gives a useful degree ofdetection limit.

In some embodiments there is provided a method of producing an isolateof the type AG compounds from honey by obtaining a honey and thenprocessing the honey by steps selected from: filtration,ultra-filtration, reverse osmosis, solvent extraction, precipitation, orcombinations thereof and collecting a high molecular weight isolate fromthe processing step.

In some embodiments, the honey or honey analogue may be filtered and thehigh molecular weight fraction collected so as to increase theconcentration of AG in the high molecular weight isolate or to removethe high molecular weight isolate to reduce the AG concentration in theresidual honey, in some embodiments, honey may be filtered to obtain ahigh molecular weight fraction via a 10 kDa filter. In some alternativeembodiments, the filter size may be via a 20 kDa filter. In somealternative embodiments, the filter size may be via a 30 kDa filter.

Examples of varying topical ailments to which the formulations describedabove may be applied include wounds, burns, skin irritations, acne,pustules, skin grafts, ulcers and combinations thereof.

As should be appreciated, the above recognises the importance of asingle group of compounds being type AG compounds in honey and theirinfluence on an immune stimulation phase. The application teaches of anisolate and related AG products that may then be used for a variety ofmethods and uses. An advantage of product and methods and uses are thatnatural variations are limited or avoided making the end product moreconsistent in line with that critical for pharmaceutical/medicalapplications. In addition, the knowledge around type II AG may be usedto tailor medical products for desired applications unlike having torely totally on nature.

While the above embodiments have been presented separately, it should beappreciated that this is not limiting and aspects of one of the aboveembodiments may also be applied to other embodiments.

WORKING EXAMPLES

The compositions and methods are now described with reference toexamples illustrating embodiments of the composition.

Example 1

In this example, a honey based mixture was formed that had been dilutedto negate the sugar effects on microbial survival so thatimmunostimulatory effects could be observed.

Production of primed macrophage cells i.e. those that produce TNFα werecompleted by a WST-1 assay to measure the toxic levels of honey samples.

The method was completed as follows.

Artificial honey was prepared by dissolving fructose (192 mg), glucose(180 mg) and sucrose (4 mg) (all three from Sigma Aldrich) in RPMImedium (10 mL) to simulate a 4% (w/v) honey concentration (where theundiluted honey contains 48% fructose, 45% of glucose, 1% of sucrose),filtered by passing through 0.20 μm (DISMIC-13CP) disposable Milliporefilter and stored at 4° C. THP-1 cells were harvested, centrifuged andresuspended in RPMI medium giving a concentration of 1×10⁸ cells/ml. Thecells were then placed in a 96-well plate (Greiner) at 50 μL/well (5×10⁴cells/well) and 4, 2, 1, 0.5, 0.25, 0.125, 0.06, 0.03 and 0.15% (w/v)honey solutions (50.0 μL) were added, resulting in 2, 1, 0.5, 0.25,0.125, 0.06, 0.03, 0.15 and 0.007% (w/v) final concentrations of honeyin 100 μL final volume and incubated for 24 h at 37° C. (Section 2.3.4).The control wells without cells were filled with the RPMI medium. Then10 μL/well of cell proliferation reagent (WST-1) was added to the cells(i.e. 1:10 dilution) and mixed thoroughly on a shaker for 1 min and theplate was incubated at 37° C. for 1 h. The absorbance of the sampleswere measured against a background control as blank using a micro plate(ELISA) reader at 450 nm set at a reference wavelength of 620 nm.

The negative control was monocytic, leukemia cells with RPMI medium, andpositive control Camptothecin which was used at different concentrations(10 uM, 1 uM, 0.1 uM) to kill the cells.

The results show the effect of controls (FIG. 2) and 22 honey samples(FIG. 3) on cells after 1 hour following the addition of the WST-1reagent. The artificial honey solution was showing variance on eachplate due to the assay. So, the sugar control was standardized againstthe negative control. Then the effects of the honeys across multipleplates were also standardized against the effects of the artificialhoney sugar solution, to remove plate-to-plate variations.

It was observed that the highest honey concentrations tested, 2 and 1%(w/v) honey, reduced cell metabolic activity by as much as 60% of thecontrols. Thus, it was concluded that both 0.5% and 0.25% concentrationsof all honey samples were the highest non-toxic doses that could beused. This was confirmed by re-testing the honeys at 0.5% (w/v), thehighest non-toxic concentration.

From the above results it is concluded that some honeys can still showup to 20% toxicity, while some honeys are completely non-toxic effectsat 0.5% concentration. In the interests of using the greatest commonconcentration for multiple honeys for ease of testing, it was decidedappropriate to use one concentration for all honey samples. Theconcentration of 0.5% (w/v) honey is approximately 50 mg/ml was to usefor further examples as this allowed cells to grow at a moderate ratewithout an immediate kill effect.

Macrophage cells were then matured to a point where they were primed andready to respond to an input. Adding varying levels of LPS and measuringthe cytokine TNFα response confirmed the maturity and readiness of themacrophage cells. A standard dose response was found i.e. the larger theLPS dose, the larger the cytokine response.

A variety of different honeys were then added to the primed macrophagecells and the cytokine response measured to determine if there were anytrends. Methylglyoxal (MGO) was also added with or without honey todetermine if this ingredient of manuka honey also influenced the immunestimulation effects. A sugar solution was also used to determine if thishad any effect as well.

The results found are shown in FIG. 4 where INT-01=0.5 year oldnorthland manuka honey; INT-03=5 year old northland manuka honey;INT-12=0.3 year old east coast manuka honey; INT-15=1.5 year oldnorthland kanuka honey; INT-16=2.5 year old Waikato manuka honey;INT-19=1.5 year old unknown source manuka honey; INT-20=1 year old southisland clover honey.

As illustrated, both MGO and sugar atone had no impact on immunestimulation and instead another component of honey present in the manukaand kanuka samples tested was ac responsible for the pro-inflammatoryresponse.

Some art suggests the presence of LPS in honey may contribute to theobserved immune stimulation response. The honey's tested by theapplicants were analysed for LPS content however once dilutions weretaken into consideration, the confounding effect of LPS is minimal witha maximum of 1 ng LPS being detected in the samples, being vastly lessthan that required to establish the response observed in the LPS doseresponse tests completed above.

In conclusion, the pro-inflammatory effects observed, particularly fromkanuka and manuka honeys must be due to a compound(s) within these honeyand is not attributable to art compounds such as sugar content, MGOcontent or LPS content in the honey.

Example 2

Heating honey is a practice employed by some beekeepers. Heating manukahoney in particular has been found to increase the MGO content (andprice) of the honey although to the detriment of other compounds in thehoney. Heat is also associated with the production of contaminatingcompounds in honey as well. Heat is also well known to break downcertain compounds and reduce activity.

The effect of heat on the pro-inflammatory response noted in Example wastested.

Honey samples (5% (w/v) and LPS (1000 ng/mL) were prepared in water andheated at 80° C. for 30 min in a shaking water bath. After heatprocessing, further dilutions were performed in RPMI medium and theresulting honey tested for cytokine response.

The results were highly compelling in that the rate of cytokineproduction decreased by approximately 40% in samples that had beenheated. This result shows that the compound(s) responsible for theobserved pro-inflammatory response are somewhat heat sensitive but havesome resistance to heat as well in view of this observation, compound(s)were identified as not being more sensitive protein or enzymatic activesbut instead more likely to be one or more larger chemical compounds thatare at least in part chemically modified by heat.

Example 3

In this example, honey found to have pro-inflammatory activity Example 1were filtered to 10 kDa and the resulting filtrate (less than 10 kDa)tested to determine if the compound(s) responsible for thepro-inflammatory effects were still present or not. The filtrate testednegative for pro-inflammatory response hence the active compound(s) musthave been larger sized (greater than 10 kDa) being of a high molecularweight size.

Example 4

In this example, the pro-inflammatory response honey samples wereirradiated to determine if the effects were influenced by irradiation.Irradiation is known in the art to reduce activity of honey,particularly where the activity is due to larger chemical compoundsand/or biologically active compounds in the honey. The trial identifiedan approximately 30% reduction in pro-inflammatory effect betweensamples without irradiation and those with irradiation. This result isin line with that observed for heat in Example 2 where thepro-inflammatory response reduces although not totally supporting thefact that the compound(s) responsible have some stability and are notsensitive compounds such as active enzymes.

Example 5

In this example, the amount and nature of the high molecular weight copound(s) were determined.

Method

Five honey's were tested being labelled H01=0.5 year old Northlandmanuka honey, H03=5 year old Northland manuka honey; H15=1.5 year oldNorthland kanuka honey, H16=2.5 year old Waikato manuka honey and H20=1year old South island clover honey.

Each honey was separated using centrifugal ultrafilters (10 kDamolecular weight cut-off) and the retentate collected being the 10 kDaor greater fraction (termed hereafter as the high molecular weight (HMW)fraction.

Retained monosaccharides, present in the HMW fractions, were analysed byhigh-performance anion-exchange chromatography (HPAEC).

Neutral constituent sugar compositions of the HMW fractions weredetermined by gas chromatography-mass spectrometry (GC-MS) of alditolacetate derivatives after hydrolysis of the polysaccharides present totheir component monosaccharides. Identifications were based on peakretention times and by comparison of electron impact mass spectra withstandard spectra.

Glycosyl linkage compositions were determined by GC-MS of partiallymethylated alditol acetate derivatives. Each sample was analysed induplicate.

High molecular weight honey fractions (0.25 mg) were methylated usingNaOH and CH₃I in DMSO. The methylated polysaccharides were hydrolysedwith 2.5 M TFA (200 μL, 1 h, 121° C.), concentrated to dryness under anair stream at 40° C., then reduced with 1.0 M NaBD₄ in 2.0 M NH₄OH (200μL) overnight at 25° C. The reaction was stopped by the addition ofglacial acetic acid (50 μL). Borate was removed as volatiletrimethylborate by addition of 5% v/v acetic acid in MeOH (3×0.5 mL),and concentrating under an air stream at 40° C., followed by addition ofMeOH (3×0.5 ml) and concentrating to dryness under an air stream at 40°C. The resulting alditols were acetylated in acetic anhydride (100 μL)and TFA (100 μL) for 30 min at 50° C. and extracted into CH₂Cl₂ foranalysis. The partially methylated alditol acetate derivatives wereseparated by GC on an Agilent HP-5MS fused silica capillary column andanalysed by MS using a Hewlett Packard 5973 MSD. Identifications werebased on peak retention times and by comparison of electron impact massspectra with standard spectra. Linkage compositions are expressed asmole percent of the total linkages detected.

The HMW fractions were dissolved in (deuterium oxide) (0.6 mL) andtransferred to 5 mm NMR tubes. NMR spectra were recorded on a Bruker 500MHz spectrometer at 30° C.

The size-exclusion (SEC) system consisted of a Waters 2690 Allianceseparations module, a Waters 450 variable wavelength detector set at 280nm and a Waters 2410 refractive index monitor. Samples (1 mg mL-1) werecentrifuged (13,500 rpm, 5 min) before injection (100 μL) and elutedwith 0.1 M LiNO₃ containing 0.02% NaN₃ (0.7 mL min-1) from two columns(TSK-Gel G5000PWXL and G4000PWXL, 300×7.8 mm, Tosoh Corp., Tokyo, Japan)connected in series. Molecular weights were estimated by comparison ofthe peak elution volumes with those of pullulan standards.

Results

Fractionation of the honey samples resulted in yields of the >10K MWmaterial of between 1.5 and 4.5 mg, representing 0.03% and 0.06% of theoriginal honey.

Honey samples H16 and H20 included considerable amounts of glucose andfructose as retained by the ultrafiltration membranes. This is reflectedin both the higher amount of glucose detected in the constituent sugaranalysis and sharp signals in the NMR spectra of these samples. Theother samples contained much lower amounts of free monosaccharides.

Neutral constituent sugar analysis showed that the HMW fractions of thehoney samples contained between 14 and 27% w/v carbohydrate (Table 1).The analyses showed the presence of arabinose and galactose in thefractionated honey samples. These sugars were present in higher amountsin the manuka and kanuka honeys, than in the clover honey. The presenceof glucose probably reflects the presence of free glucose, as determinedby HPAEC.

Detection of mannose (as mannitol hexaacetate) in the samples may befrom glycoproteins present in the high molecular weight fractions of thehoneys, or could result from the reduction of free fructose in thesamples to mannitol and glucitol during the preparation of the alditolacetates.

(Note: sample H01 was analysed separately from the other samples and thehigher arabinose and galactose contents observed may be due todifferences obtained in standard sugar response factors in thisanalysis.)

TABLE 1 Constituent sugar composition of the high molecular weightfractions of honey samples analysed using the reductive hydrolysismethod. Constituent sugars (dry weight %)* Sample Ara Man Glc Gal TotalH01 7.4 2.8 1.4 10.8 22.4 H03 4.1 1.5 2.0 6.4 14.0 H15 4.5 1.5 0.5 8.414.9 H16 4.9 2.4 10.7 9.1 27.1 H20 0.9 3.2 10.3 2.6 17.0 *Values are theaverages of duplicate analyses

Glycosyl linkage analysis revealed the presence of arabinose andgalactose in the fractions indicating the presence of arabinogalactan(AG).

Type 1 AGs are usually found as neutral side-chains on plant cell-wallpectic polysaccharides, while Type II AGs are often present asAG-proteins (AGPs), rich in hydroxyproline.

In order to determine whether Type 1 or Type II AGs were present in thehoneys, the glycosyl linkage compositions of the samples were determined(Table 2).

The glycosyl linkage analyses of the manuka and kanuka honeys (H01, H03,H15 and H16) were consistent with polymers comprised of a highlybranched backbone of 1,3-linked Galp residues, with side-chains made upof Araf-containing oligosaccharides, typical of Type II AGs Theselinkages represented 7-10 of the total weight of the HMW fractions fromthe manuka and kanuka honeys, which was consistent with, but slightlylower than, that calculated from the sum of arabinose and galactose inthe constituent sugars analyses (Table 1).

The glycosyl linkage analysis of the clover honey also showed thepresence of Type II AGs, but in much lower amounts (1.3% of the weightof the HMW fraction, compared with 3.5% from the sum of arabinose andgalactose).

TABLE 2 Glycosyl linkage composition of the high molecular weightfractions of honey samples analysed by GC-MS of partially methylatedalditol acetates. Linkage composition (mol %)^(a) H01 H03 H15 H16 H20Sugar linkage (9.8%^(b)) (10.0%^(b)) (12.0%^(b)) (12.0%^(b)) (4.0%^(b))Arap terminal — 1.7 0.1 0.3 — Araf terminal 27.5 24.6 30.8 31.1 12.9 2-3.4 4.2 2.1 2.3 — 3- 1.2 1.9 1.7 2.3 — 5- 4.5 6.6 5.4 8.5 6.7 Galpterminal 4.2 1.4 6.8 3.3 2.1 3- 8.0 4.8 9.9 7.1 4.7 6- 5.1 5.9 4.1 2.42.1 3,6- 27.5 27.1 23.8 22.5 7.6 3,4,6- 1.3 — 5.4 3.1 — Glcp terminal7.5 6.7 4.7 7.2 35.9 Manp 2- 4.8 3.3 3.2 3.7 15.3 3,6- 1.4 1.0 0.9 0.75.4 Other 3.6 10.8 1.1 5.8 7.3 Total 100 100 100 100 100 ^(a)Values arethe averages of duplicate analyses ^(b)Values in parentheses are %carbohydrate content calculated from sum of all linkages, compared tointernal standard of myo-inositol (10 g)

The 1H NMR spectra of HMW honey fractions revealed in the protein NMRspectroscopy are shown in FIGS. 5A to 5E. The spectra for H16 (FIG. 5D)and H20 (FIG. 5E), that contained high amounts of free sugars showedsharp signals at about 5.21 and 4.61 ppm, that were attributed to H-1 ofand -Glc, respectively. Samples H01 (FIG. 5A). H03 (FIG. 5B), H15, (FIG.5C) and H16 snowed H-1 signals at 5.23 and 5.08 ppm, with H15 and H16showing an addition H-1 signal at 5.43 ppm. These H-1 signals wereconsistent with NMR assignments of -L-Araf residues of AGs. Severalother H-1 signals were observed in 4.4-4.6 ppm range that were assignedto H-1 of -D-Galp residues. These data provide further evidence for thepresence of Type AGs, probably present as AGPs.

The HMW fractions of the honeys showed complex chromatograms on sizeexclusion chromatography (SEC) eluting from the V₀ to the V_(t) of thecolumns (FIG. 6). Sample H01 showed a predominate peak (˜18.5 mL)eluting just before the 24 kDa pullulan standard and a second smallerpeak (˜16.7 mL) eluting just after the 100 kDa pullulan. Similar peakswere also observed for H03, but in smaller amounts. H15, H16 and H20also showed peaks eluting at ˜18.5 and ˜16.7 mL (24 kDa and 100 kDa,respectively), but H15 also showed a peak at 20 mL, eluting after the 24kDa standard. Additionally H16 and H20 showed peaks at 21-22 mL, assumedto be due to low molecular weight sugars present in these samples (seeTable 2).

Conclusions

Size fractionation of five honey samples analysed with 10 kDacentrifugal filters gave high molecular weight (HMW) tractions thatrepresented 0.03-0.09% by weight of the total honeys. The carbohydratecontent of these HMW fractions was between 14 and 27% w/w andconstituent sugar analysis showed that in four samples (manuka: H01, H03kanuka: H15, H16) arabinose and galactose accounted for the majority ofthis carbohydrate. In sample H20 (clover), these sugars accounted foronly about 20% of total carbohydrate.

Glycosyl linkage and NMR spectroscopic analysis showed that the honeyscontained type II arabinogalactans (AGs), that are often present asarabinogalactan proteins (AGPs). The basic structure is shown in FIG. 1.

Based on the yields of the HMW fractions and their sugar compositions,it is estimated that the type II AG content of the honey samples isapproximately:

-   -   H01: 0.007%    -   H03: 0.009%    -   H15: 0.008%    -   H16: 0.004%    -   H20: 0.002%

Example 6

The above finding the arabinogalactans are the compounds present in thehigh molecular weight fraction suggests that these compounds areresponsible for the pro-inflammatory response observed in earlierexamples. This trial tested this hypothesis by testing thepro-inflammatory response of the controls being cells and honey andcells and LPS against varying levels and concentrations ofarabinogalactans both with and without honey.

The results are shown in FIG. 7.

The results show that arabinogalactans clearly are responsible for thepro-inflammatory effect observed in certain honey. Samples that hadarabinogalactans had a far greater pro-inflammatory response thansamples without. The arabinogalactans could be used either on its own(not type II) and still gave a positive pro-inflammatory effect or couldbe ‘spiked’ into honey to give a great pro-inflammatory effect. Thisresult shows that it possible to produce honey based dressings thatinclude tailored levels of arabinogalactans which may be used to tailorthe pro-inflammatory effects of the dressing for example by adding morearabinogalactans to increase the effect and conversely, removearabinogalactans to reduce the pro-inflammatory effect.

Example 7

In this example the ability of three New Zealand honeys to elicit therelease of TNF-α from the monocytic cell lines THP-1 and U937 wereinvestigated and the bioactive component responsible identified.

The study identified that kanuka, manuka, and clover honeys eachstimulated TNF-α release from PMA-differentiated THP-1 cells. Kanukahoney gave the most potent inflammatory effects as illustrated in FIG. 8although all honeys tested (manuka and clover as well) exhibited somedegree of inflammatory effects. A honey analogue was also tested thatgave no significant pro-inflammatory response. Further, a pure MGOsample was also tested which also gave no significant pro-inflammatoryresponse. The results particularly for MGO demonstrate that thepro-inflammatory effect is not correlated to MGO content in honey or theanti-microbial activity of a honey as measured by UMF activity ornon-peroxide activity.

The major immunostimulatory activity of kanuka honey was associated witha high molecular weight (>30 kDa) component that was inhibited bypolymyxinB B and partially heat-labile. FIG. 9 illustrates the resultsfound for fractions of kanuka honey showing how the active compound isof a comparatively high molecular weight.

LPS levels in the honeys did not adequately explain the level ofimmunostimulatory activity suggesting that an additional factor waspresent. The contribution of a type II arabinogalactan protein (AGP)previously identified in kanuka honey was tested. The kanuka AGPstimulated the release of TNF-□□ from THP-1 and U937 cells, and appearsto be largely responsible for the activity of kanuka honey.

Example 8

In this example, the effect or otherwise of pure AGP was analysed todetermine how AGP influences cytokine stimulation as exemplified byTNF-a production from THP-1 cell line.

The results found are shown in FIG. 10.

As shown in FIG. 10, a honey analogue without AG produces noinflammatory effects. AGP isolate as expected produces a measurableinflammatory effect. When this isolate was added to the honey analogue,a pro-inflammatory effect resulted as expected.

Trials were completed adding polymyxinB (PmB) to an LPS and AGP isolatecomposition to confirm the negative effects of polymyxinB oninflammation.

Also tested was a combination of kanuka honey tested as per earlierExamples above but where the kanuka was fortified with the AGP isolate.The resulting impact on cytokine production/inflammation was above thatpredicted and more than the anticipated additive effect as shown in FIG.10.

Example 9

In this example the dose response of AGP isolate was tested and comparedto a standard, LPS in terms of pro-inflammatory effects.

Varying doses of LPS were tested against THP-1 cell line and thecytokine response measured. An expected linear response was obtained.Subsequent analysis using AGP isolate was then tested and a similarlinear response was obtained. Results are shown in FIG. 11 indicatingthat the immunostimulatory effects appear to transition with a dose ofapproximately 5 μg/g. This dose or higher doses e.g. 10 μg/g cause ameasurable immunostimulatory effect on monocytes while a lower levele.g. less than 1 μg/g result in no or minimal effect on monocytes. Theresults further reinforce the presence of type II AG compounds and theirinfluence on inflammation.

Example 10

Further characterisation work was completed to determine the make up ofthe AG compound in the honey.

Fractionation of manuka, kanuka and clover honeys indicated the >10 kDafraction contained small amounts of type II arabinogalactans (AGs),which are often present as arabinogalactan proteins (AGPs). AGPs wereisolated from the >10 kDa fraction of kanuka honey using glucosyl Yarivreagent and their composition and structure analysed. Constituent sugar,glycosyl linkage and NMR spectroscopy analysis of the purified AGPfraction revealed a predominance of neutral sugars, mainly galactose andarabinose, linked in a highly-branched structure typical of type II AGs.The molecular weight of the major component of the purified AGPs was˜110 kDa, as determined by size-exclusion chromatography-multi-anglelaser light scattering (SEC-MALLS). The Yariv supernatant fractioncontained less total sugar, especially galactose, and more protein thanpurified AGPs. Linkage analysis indicated this fraction also containedan AG-type polymer in addition to various other polysaccharides andSEC-MALLS indicated the molecular weight of the major component was ˜32kDa.

Example 11

As noted above, isolates may be produced containing type it AG derivedfrom honey.

One method for obtaining such isolates is to separate the type II AG'sby using centrifugal ultrafilters (10 to 30 kDa molecular weightcut-off) and the retentate collected being the 10 kDa or greaterfraction, termed hereafter as the high molecular weight (HMW) fraction.

Other apparatus capable of 10 to 30 KDa filtration may be used includingfiltration, ultrafiltration, reverse osmosis, centrifugation andcombinations of these process operations.

Example 12

A more detailed protocol used to isolate type II AGP from honey isdescribed.

The protocol comprised three main steps being:

-   -   Ultrafiltration with a 10 kDa filter,    -   Subsequent removal of some proteins by salt precipitation and    -   Subsequent precipitation of AGP with Yariv reagent.

As may be appreciated, steps after the initial ultrafiltration stepabove may be omitted if it is not necessary for the isolate to containhighly purified AGP.

Example 13

Examples of varying topical formulations are now provided in Table 1below.

TABLE 3 Example Topical Formulations Formulation Number Components 1 Adressing formed from alginate fibre, honey impregnated in the fibre,type II AG isolate mixed into the honey 2 A dressing formed from cottongauze fibre, honey impregnated in the fibre, type II AG isolate mixedinto the honey 3 A gel or putty or sheet made from alginate particlesmixed with honey and type II AG isolate 4 A gel or putty or sheet madefrom carboxymethylcellulose fibre, honey partly to fully impregnated inthe fibre, type II AG isolate mixed into the honey 5 A gel, putty,sheet, cream or ointment made from honey, ethoxylated oil, myristylmyristate or other wax materials such as beeswax and type II AG isolatemixed together 6 A gel, putty, sheet, cream or ointment made from honey,caprylyl capryl glucoside, myristyl myristate or other wax materialssuch as beeswax and type II AG isolate mixed together 7 Honey analoguefortified with type II AG compounds and mixed into a cream base.

Example 14

As noted above, it is also possible to tailor a formulation by testingsamples and then selecting samples with either high or low type II AGcontent and performing subsequent blending steps to either maximise typeII AG content in the formulation or instead to minimise type II AGcontent.

In this example a series of samples are tested and tailored by selectionand blending to achieve a desired maximising or minimising.

Ten batches of honey are received at a honey processing premises andsamples taken from each honey batch. The samples are analysed todetermine the content or otherwise of type II AG in each sample.

Samples with the greatest levels of type II AG are separated and blendedtogether to form a honey with a higher level of inflammatory action. Theblended honey may be used to form an isolate by subsequently subjectingthe blend to filtration to remove the type II AG compounds.

Samples with lower levels of type II AG or where AG is not present maybe selected and blended separately to form a honey with minimalinflammatory action. The blended honey may be used to in medicalapplications where inflammatory effects are to be avoided such as on asensitive or open wound or, if the blend has a high MGO content, may beused to instigate and anti-microbial challenge to the wound or otherskin ailment but minimise inflammation at the skin site.

Example 15

A detection protocol is now described with which to complete the testingdescribed above.

Antibodies JIM 4, JIM, 13, JIM 14, JIM 15, MAC 207, LM 2 and LM 14 weretested. All were useful tar an AG-ELISA but JIM 13 yielded significantlybetter results than the others, having the highest binding capacity.

Sandwich ELISA's were also tested combining the abovementionedantibodies with a mouse capture antibody for AG. Sandwich ELISA was alsosuccessful.

In practice, the protocol found useful is to first bind antibody e.g.JIM13 to the AG compounds and then detect bound JIM13 via a secondaryantibody such as an anti-rat secondary antibody. From the result, the AGconcentration may be derived.

The method described can detect AG concentrations down to 0.5 μg/g andprovides linear results between 0.5 and 5 μg/g AG in the samplesolution.

Example 16

As noted above it is possible to select and breed plants to influencethe content of type II AG in honey produced from the plants.

One method of selection and breeding to increase the content of type IIAG compounds in a honey is to test a range of plants ideally from asingle species known to already contain significant amounts of AGcompounds e.g. kanuka plants, and select for varieties that produce thegreatest concentrations of type II AG's in the plant nectar. The plantsmay then be cross-bred with other plant varieties to form ‘super’ typeII AG producing plants. Honey is then produced via bees or artificiallyfrom the bred plants and in turn high type II AG honey may bemanufactured.

The opposite may also be the case where a low AG content honey is to beproduced. Tests may be completed on a range of low AG containing honeyse.g. clover and varieties bred to minimise the AG content and honeyproduced from these minimised varieties.

Example 17

Breeding and selection methods are described above. An initial screeningmethod may further include selection of plants that provide a challengefor the bee to extract the plant nectar.

Using kanuka honey as an example, kanuka honey is thought to containmore type II AG, because kanuka flowers are narrower than manukaflowers, thereby requiring the bee to extract nectar with greaterphysical force thereby taking up more AG.

Using this finding, specific breeding for narrow-flowered plants shouldincrease type II AG concentration (as long as the bees are able toaccess the nectar).

Aspects of the compositions and methods have been described by way ofexample only and it should be appreciated that modifications andadditions may be made thereto without departing from the scope of theclaims herein.

What we claim is:
 1. Honey fortified with an isolate containing type IIarabinogalactan wherein the average molecular weight of thearabinogalactan is 40,000 to 1,100,000 daltons and wherein the ratio ofarabinose to galactose ranges from 0.1-2.0 parts arabinose to 1.0 partsgalactose.
 2. The honey as claimed in claim 1, wherein the honeycontains at least 5 μg/g type II arabinogalactan.
 3. The honey asclaimed in claim 1, wherein the honey contains at least 10 μg/g type IIarabinogalactan.
 4. A composition formulated for topical application toa body area of a patient in need thereof containing honey fortified withan isolate containing type II arabinogalactan wherein the averagemolecular weight of the arabinogalactan is 40,000 to 1,100,000 daltonsand wherein the ratio of arabinose to galactose ranges from 0.1-2.0parts arabinose to 1.0 parts galactose.
 5. The composition as claimed inclaim 4, wherein the composition is selected from a dressing, a cream,an ointment, a gel, and combinations thereof.
 6. The composition asclaimed in claim 4 wherein the type II arabinogalactan isarabinogalactan protein.
 7. A method of stimulating the immune system ofa patient in need thereof by topically applying said honey of claim 1 tosaid patient.
 8. The method of claim 7 wherein the honey is formulatedfor application to a recalcitrant topical body area on a patient.
 9. Amethod of treatment of a recalcitrant skin condition on a topical bodyarea on a subject in need thereof by: a. applying said honey of claim 1initially, and, as healing progresses; b. applying a honey-basedcomposition with a quantity of type II arabinogalactan compounds removedfrom the honey-based composition.
 10. The method as claimed in claim 9,wherein the quantity of type II arabinogalactan removed from thehoney-based composition is such that the honey-based composition hasless than 5 μg/g type II arabinogalactan.
 11. The method as claimed inclaim 9, wherein the quantity of type II arabinogalactan removed fromthe honey-based composition is such that the honey-based composition hasless than 1 μg/g type II arabinogalactan.
 12. A method of treatment of asensitive skin condition on a topical body area on a subject in needthereof by: a. applying a honey-based composition with a quantity oftype II arabinogalactan compounds removed from the honey-basedcomposition, and, as skin healing progresses; b. applying said honey ofclaim
 1. 13. The method as claimed in claim 12, wherein the quantity oftype II arabinogalactan removed from the honey-based composition is suchthat the honey-based composition has less than 5 μg/g type IIarabinogalactan.
 14. The method as claimed in claim 12, wherein thequantity of type II arabinogalactan removed from the honey-basedcomposition is such that the honey-based composition has less than 1μg/g type II arabinogalactan.